Extraction of VDE was achieved according to the
protocol mentioned in references
[17,19]
, with some
modi
fi
cations. The chloroplast suspension (1 ml) containing
thylakoid membranes was washed twice with 2 ml of 50 mM
Tris
–
HCl buffer (pH 7.5)/1 mM MgCl
2
(1:1, v/v) by
centrifugation at 17,420
g
for 30 min. The washed
thylakoid membranes were suspended in 1 ml of the same
buffer and sonicated with a 1210R-DTH Branson UltrasonicCleaner (Danbury, USA) 20 times for 12 s at 10 s interval.
The suspension was centrifuged at 27,220
g
for 90 min.
The supernatant was designated the VDE extract.